A major hurdle in the development of effective vaccines against pathogens that reside within macrophages, including Mycobacterium tuberculosis, is how to deliver antigens in a manner that stimulates a protective cellular immune response. Recent investigations involving antisense mutants of M. tuberculosis that have diminished production of iron-cofactored superoxide dismutase (SOD) show that they are attenuated, induce strong CD4+ and CD8+ T-cell responses in mice, and exhibit promising activity as a vaccine prototype. These effects appear to be related to an unmasking of the innate immune responses normally inhibited by SOD, which is a prominent extracellular enzyme of M. tuberculosis and other pathogenic mycobacteria. The enhanced innate host immune responses presumably permit apoptosis-associated cross-presentation of microbial antigens via MHC Class I pathways to induce strong adaptive CD4+ and CD8+ T-cell responses, in contrast to the current vaccine for tuberculosis, BCG, which exhibits a predominant CD4+ T-cell response and minimal CD8+ T-cell responses. The goals of the current proposal are first, to characterize the cellular and cytokine responses in the lung observed early after infection with SOD-diminished M. tuberculosis, as rapid pulmonary interstitial infiltration with mononuclear cells undergoing apoptosis appears to be a process unique to the SOD-diminished strains that is not observed during infection with either virulent M. tuberculosis or BCG. This should define the conditions under which antigen cross-presentation occurs in vivo, yielding information that may be useful for a variety of vaccines. The second goal is to construct non-reverting SOD-diminished mutants of H37Rv and BCG by replacing the wild-type SOD allele with mutant alleles, some of which encode enzymatically less efficient mutants of SOD. This should yield a SOD-diminished vaccine candidate that is stable and safe enough for administration to man. The third goal is to determine the optimal level of SOD production for maximal vaccine efficacy and the immune correlates of protection. Diminishing the production of factors produced by intracellular pathogens that inhibit macrophage apoptosis is a strategy for making new vaccines that achieve MHC Class I antigen presentation. This should have implications not only for tuberculosis but also for other infectious diseases in which CD8+ T-cell responses are a critical component of a protective immune response.